The enzyme ACC synthase is essential to the production of ethylene in higher plants. It is well known that ethylene is related to various events in plant growth and development including fruit ripening, seed germination, abscission, and leaf and flower senescence. Ethylene production is strictly regulated by the plant and is induced by a variety of external factors known as ethylene-inducible events. These include the application of auxins, wounding, anaerobic conditions, viral infection, elicitor treatment, chilling, drought, and exposure to ions such as cadmium and lithium ions. In addition, it recently has been shown that ethylene production begins after harvest (Tian et al., J. Amer. Soc. Hort. Sci., 119:276-281 (1994)).
The pathway for ethylene synthesis in plants was first described by Adams and Yang, PNAS, U.S.A., 76:170-174 (1979) who identified 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. The physiology and biochemistry of ethylene synthesis was extensively reviewed by Yang and Hoffman in Ann. Rev. Plant Physiol., 35:155-189 (1984). In the ethylene biosynthetic pathway, methionine is catalyzed by the enzyme S-adenosylmethionine synthetase to form S-adenosylmethionine (SAM). SAM is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase. This three-membered-ring amino acid is then metabolized to yield ethylene, a reaction catalyzed by the enzyme ACC oxidase.
The ethylene-forming enzyme genes in tomato were the first to be isolated. Smith et al., Planta, 168:94-100 (1986) reported the rapid appearance of an mRNA correlated with ethylene synthesis, which encodes a protein of molecular weight 35,000. This formed the basis for the development of a number of molecular strategies to inhibit ethylene formation in certain transgenic plants. One such method is based on antisense RNA.
As is well known, a cell manufactures protein by transcribing the DNA of the gene encoding that protein to produce RNA, which is then processed to messenger RNA (mRNA) (e.g., by the removal of introns) and finally translated by ribosomes into protein. This process may be inhibited by the presence in the cell of "antisense RNA." The term antisense RNA means an RNA sequence which is complementary to a sequence of bases in the mRNA in question in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, thus preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. This antisense RNA may be produced in the cell by transformation of the cell with an appropriate DNA construct arranged to transcribe the non-template strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).
The use of anti-sense RNA to downregulate the expression of specific plant genes is well known. Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference, e.g., lack of anthocyanin production in flower petals of petunia leading to colorless instead of colored petals (van der Krol et al., Nature, 333:866-869 (1988)); or at a more subtle biochemical level, e.g., change in the amount of polygalacturonase and reduction in depolymerization of pectin during tomato fruit ripening (Smith et al., Nature, 334:724-726 (1988)).
Another more recently described method of inhibiting gene expression in transgenic plants is the use of sense RNA transcribed from an exogenous template to downregulate the expression of specific plant genes (Jorgensen, Keystone Symposium "Improved Crop and Plant Products through Biotechnology," Abstract X1-022 (1994)). Thus, both antisense and sense RNA have been proven to be useful in achieving downregulation of gene expression in plants.